Effects of point mutation C→T at - 64 of human δ globin gene promoter on DNA binding proteins

By electrophoretic mobility shift assay (EMSA), the effect of point mutation C→T at - 64 of human δ-globin gene on its binding proteins has been studied. Two segments of 36 bp from - 83- - 48 bp of the 6 globin gene promoter, named WOG and MOG, were synthesized. WOG includes wild type CAAT-like box (CCAAC), while MOG includes the mutant CAAT-like box (CCAAT, -64 C→T). Results indicate that: ( i ) in erythroid cell lines MEL, K562 and Hemin induced K562, the affinity of MOG with CCAAT binding protein (CBF) and GATA-1 was greatly increased; ( ii ) in Hemin induced K562 cell line, there were another two novel specific DNA binding proteins, named C and D temporarily, besides the above two factors. The former was combined with WOG and MOG, likely indicating its relation with the increased gene expression after induction. The latter was only combined with MOG, which had possible relationship with the point mutation of - 64 C→T; ( iii ) EMSA also indicates that the suppression mechanisms of the expression of 6 globin gene is different in various periods of human developments. The result evidently supports the hypothesis that the defect CAAT-like box in human 6 globin gene contributes the main reason of its low level expression. The defect c/s-acting element CAAT-like box affects gene expression by its combination with the frans-acting element CBF and GATA-1.     

Mouse neural stem cells culturedin vitro and expressing an exogenous gene

Neural stem cells are the multipotential, self-renewing cells in central nerve system, and play an essential role in the development and differentiation of nerve system. Neural stem cells can be used to treat the nerve system diseases, especially, the transplantation of neural stem cells to rescue the degenerated neural cells has become a very promising therapeutic way. We successfully cultured neural stem cells isolated from the brains of embryonic micein vitro and determined their distribution in the E17 mice brains. The neural stem cells were transfected with adenoviral vector carrying GFP (green fluorescence protein) gene and then highly expressed the exogenous gene. It paves the way for gene therapy of degenerative nerve system diseases.     

Clony color assay coupled with 5FOA negative selection greatly improves yeast three-hybrid library screening efficiency

The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.     

硝苯地平探针高效液相色谱法测定大鼠肝微粒体CYP3A酶活性

以硝苯地平为探针药物,研究建立一种大鼠肝微粒体CYP3A酶活性定量测定的高效液相色谱方法,并优化色谱条件和肝微粒体样品预处理方法.在测定条件下,硝苯地平经CYP3A酶代谢转化为氧化硝苯地平,其色谱保留时间7.09min,无内源性物质干扰.方法的线性检测范围1.0～40.0μmol/L,定量下限1.0μmol/L,回收率94.03%～98.88%,日内、日间相对标准偏差均小于7.3%.方法学验证结果表明,该方法快速、灵敏、准确,具有良好的专属性、精密度和稳定性,适于生物样品中氧化硝苯地平的定量测定,可用于CYP3A酶活性评价及酶动力学研究.     

氟苯尼考残留的酶联免疫检测方法的研究

对氟苯尼考进行结构改造制备半抗原及抗原,免疫家兔得到多克隆抗体。经测定,该抗体对氟苯尼考和氟苯尼考胺均有反应,且灵敏度较高,效价达到1∶480 000;在此基础上通过对各实验条件参数的优化建立了间接竞争酶联免疫检测方法,该方法检测限为0.5μg/kg,以20,50μg/kg进行空白样品添加实验,回收率为82.5%～96.0%,批间变异系数在4.1%～15.2%,可初步用于动物组织样本中氟苯尼考残留的检测.     

云南正常人群基因组健康初探

人类基因组稳定性与人类健康具有密切的联系,基因组健康水平降低,可导致神经系统、循环系统、免疫系统等多类退行性病变危险度升高。本研究采用胞质分裂阻滞微核分析（CBMN）,评价了云南正常人群淋巴细胞的基因组健康水平。研究发现,人类淋巴细胞自发微核频率随年龄增加而升高（r=0.468,p〈0.001）,且女性平均微核率显著高于男性（r=2.438,p〈0.05）。云南籍不同年龄和性别的正常人群基因组健康的基础数据结构,为基于基因组健康的个性化营养干预提供了科学依据。     

Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.     

萤火虫和海肾萤光酶在植物分子生物学中的应用

在植物分子生物学中,编码生物发光蛋白的基因作为报告基因广泛用于检测基因表达.萤火虫萤光酶和海肾萤光酶都具有定量分析方法上的优势,基于此人们开发了双萤光酶报告基因体系.本文就萤火虫和海肾萤光酶的特点及其在植物分子生物学中的应用作一概述.     

中国柞蚕肠道蛋白酶的提取与活力测定

应用丙酮沉淀法以柞蚕肠道组织为材料提取蛋白酶并进行了活力测定．这项成果为柞蚕饲养和代谢的研究提供了重要参与，并为酶在工业上的利用找到了依据．     

一种简单实用的鲎试剂检测细菌内毒素法

在鲎试剂检测内毒素原理的基础上对传统的凝胶法进行改良。经改良后的新方法与传统方法相比具有检测结果一致，鲎试剂用量少，检测时间短，操作误差小等优点。